This study assesses the effect of long-term (59 years) application of organic and inorganic fertilizers on soil organic matter and enzyme activity. Total organic C, total organic N, hot water soluble C, microbial biomass C and dehydrogenase activity were evaluated in soil from the long-term field experiment in Prague-Ruzyně (Orthic Luvisol, clay loam). Total organic C and N increased significantly in soils treated with organic fertilizers (farmyard manure, compost) and in soils with a combination of organic and mineral NPK fertilizers (manure + NPK, compost + NPK, cattle manure + straw + NPK) compared to soil treated with inorganic fertilizer, cattle slurry + straw and non-fertilized control. Farmyard manure significantly increased hot water soluble C compared to the control. Dehydrogenase activity was significantly increased by all treatments compared to control. The results indicate that additions of organic matter from various sources differ in the effects on soil organic matter and biological activity. The effect of manure was the most favourable; long-term application of cattle slurry + straw is rather similar to mineral fertilization.